EP0506377B1

EP0506377B1 - Calmodulin binding protein

Info

Publication number: EP0506377B1
Application number: EP19920302591
Authority: EP
Inventors: Ken'ichiro Hayashi; Kiyozo Asada; Takashi Hashida; Hirokazu Kotani; Ikunoshin Kato; Kenji Sobue
Original assignee: Takara Shuzo Co Ltd
Current assignee: Takara Shuzo Co Ltd
Priority date: 1991-03-29
Filing date: 1992-03-25
Publication date: 1997-12-10
Anticipated expiration: 2012-03-25
Also published as: DE69223434D1; EP0506377A3; EP0506377A2; DE69223434T2

Description

Field of industrial application:

This invention relates to a polypeptide that has the amino acid sequence of the functional domain of human caldesmon.

State of the prior art:

Caldesmon is a protein that can bind with calmodulin, actin and tropomyosin, found in all tissues except skeletal muscle and cardiac muscle; it contributes to the regulation of actomyosin system in smooth muscle. This regulatory function depends on the concentration of calcium ions (flip-flop regulation) in bringing about the regulation of the actomyosin system (Proc. Natl. Acad. Sci. USA, 78, 5652-5655, 1981). When the concentration of calcium ions is low, caldesmon binds with the actin filament-tropomyosin system, and inhibits the mutual interactions of actin and myosin. When the calcium ion concentration is increased, complexes of active calmodulin and caldesmon are formed. This complex is released from the actin filament-tropomyosin system. Because of this, the inhibition of actin-myosin interaction caused by caldesmon cease, and these interactions begin.
Caldesmon was first isolated from the smooth muscles of chicken gizzards, and it has been isolated from other vertebrates. The results of the limited digestion of chicken caldesmons by the protease α-chymotrypsin have shown that a polypeptide of the size of about 35 kDa at the carboxy-terminal end of chicken caldesmons is the functional unit that brings about flip-flop regulation that depends on the concentration of calcium ions (J. Biochem., 102, 1065-1073, 1987).
There are two isoforms of chicken caldesmon that have different molecular weights. The results of electrophoresis on sodium dodecyl sulfate-polyacrylamide gels have shown that one is of high molecular weight (120,000 to 150,000Da), now named h-caldesmon, and that the other is of low molecular weight (70,000 to 80,000Da), now named l-caldesmon. h-caldesmon is abundant in smooth-muscle tissue, and l-caldesmon is abundant in non-muscle tissue and cultured cells. The amino acid sequence of h-caldesmon and the DNA sequence that codes for it have recently been identified (Biochem. Biophys. Res. Commun [called BBRC below], 164, 503-511, 1989), as have been the amino acid sequence of l-caldesmon and the DNA sequence that codes for it (J. Biol. Chem., 266, 355-361, 1991; also Japanese Patent Application 2-37362).
Both h- and l-caldesmons are of chicken origin, and the amino acid sequence of human caldesmon is still unknown.
Human platelet caldesmon₇₇ has been purified and used to prepare antibodies to use in the domain mapping of chicken gizzard caldesmon, Journal of Biological Chemistry, 262, No. 6, 25 February 1987, 2757-2763. cDNA clones spanning a coding region of 461 amino acids of low Mγ human caldesmon have been obtained, J. CELL BIOL. 111 (5 Part 2), 163A, Abstract No 894, 1990. A cDNA probe encoding the C-terminus of avian caldesmon has been used to screen a human aorta library and clone smooth-muscle and non-muscle caldesmon-encoding cDNAs. The predictions from this are that the smooth-muscle protein is 793 aa long and the non-muscle protein, missing the central helical domain of 256 aa, is 537 aa long, Gene 112 (1992) 197-204.
The Journal of Biological Chemistry Vol. 226, No. 25, September 5th 1991, pp 16917-24 discloses the characterisation of cDNA clones encoding a human fibroblast caldesmon isoform which span the entire coding region (538 amino acids). Human, rat and chicken 1-CaD amino acid sequences are compared.
The morphological change of cells is one characteristic of carcinogenesis. The preservation of cell morphology is closely related to the actin network of the cells, and when cells are transformed, the change in cell morphology occurs in correlation with loss of actin cables, which are part of the filament system that makes up the cytoskeleton (Proc. Natl. Acad. Sci. USA, 72, 994-998, 1975). Immunohistochemical analysis have revealed that caldesmon is localized in actin cables, cell attachment sites, and membrane ruffles in normal cells, but in cancer cells, the compound is not found in any defined cell location (Proc. Natl. Acad. Sci. 81, 3133-3137, 1984).
When cultured cells are transformed with a variety of oncogenic viruses, the caldesmon concentration of the cells decreases and the phosphorylation of the caldesmon increases (Proc. Natl. Acad. Sci. USA 81, 3133-3137, 1984, and Saibo Kogaku [Cell Technology], suppl. 3, "Molecules regulating calcium signalling," 140-153, 1987).

Problems to be solved by this invention

As described above, there seems to be a close relationship between caldesmon content and the change in morphology of cells that have been transformed. If the relationship were understood, it might be possible to use human caldesmon for treatment of cancer. In addition, if the DNA sequence that codes for human caldesmon were known, it would be possible to use it for the diagnosis of cancer. Further, if it were possible to isolate a polypeptide that has activities of the actin-binding functional unit, the tropomyosin-binding functional unit, and the calmodulin-binding unit, this polypeptide could be used to inhibit or accelerate the mutual interactions between actin and myosin whatever the calcium concentration is, so that the polypeptide could be used as a vasodilator or a regulator of the movements of the digestive tract.
The purpose of this invention is to provide a polypeptide that has the activity of human caldesmon and the DNA sequence that codes for said polypeptide.

Steps taken to solve the problem.

To summarize this invention, the first part of this invention relates to a polypeptide that has a calmodulin-binding activity and an actin-binding activity characterized by said polypeptide has an amino acid sequence of sequence ID No.1 in the sequence listing in a polypeptide molecule. The second part of this invention relates to a gene that codes for the polypeptide of the first part of this invention.
The polypeptide of this invention is a polypeptide that has both calmodulin-binding activity and actin-binding activity; it is, for example, a polypeptide that has within it the amino acid sequence shown as sequence ID No.1 in the sequence listing, which sequence is that of the functional unit of human caldesmon, which unit was discovered by the inventors of this invention. Polypeptides that have within them this amino acid sequence include, for example, the polypeptides that have the sequence ID No.2 to 5 of the sequence listing.
A gene that codes for the polypeptide of artificial human caldesmon is, for example, a gene with the DNA sequence of sequence ID No.7 to 12 in the sequence listing, and genes that can hybridize to the gene with the sequence ID No.7 to 12, and which genes code for a polypeptide that has calmodulin-binding activity and actin-binding activity.
The inventors of this invention prepared a cDNA library from HeLa cells, a human cell line, and next selected cDNA clones that code for a polypeptide of human caldesmon from this cDNA library; the DNA sequence and the amino acid sequence coded for human caldesmon were deduced by DNA sequence analysis. Next, based on various finding, the inventors prepared polypeptides of various lengths, and looked for the functional domain of human caldesmon polypeptide, using calmodulin-binding activity and actin-binding activity as indices. Based on the findings described below, the inventors achieved this invention.
Below, this invention is explained in detail.
The gene that codes for human caldesmon can be isolated and analyzed as described here. To obtain the gene of human caldesmon, HeLa cells or the like can be used, and the total RNA, which includes the poly(A)⁺ RNA, of the cells, was isolated, and bound onto a cellulose carrier or the like with oligo(dT). This was used as a template in the synthesis of cDNA with reverse transcriptase. The cDNA was synthesized by the method of Okayama-Berg or by the method of Gubler-Hofmann. The cDNA synthesized in this was ligated with a plasmid or phage vector and introduced into a host, giving a cDNA library.
The cDNA library was screened for the desired clones by one of the two following methods. The amino acid sequence of a portion of the desired protein was identified, and an oligonucleotide that coded for that amino acid sequence was synthesized and used as a probe in screening. Or, an antibody that bound with the desired protein was prepared and used to screen the cDNA library constructed in an expression vector, in an immunological method.
When the immunological method of screening is used, λgtl1 is well used as the expression vector. λgt11 is commercially available; it can be purchased from, for example, Stratagene. When λgt11 is used in immunological screening, first, the library is amplified on agar plates, and nitrocellulose or nylon filters treated with isopropyl-β-D-galactopyranoside (IPTG) are placed on these plates and expression is induced, after which the protein being expressed adsorbes onto the filter. The filter is immersed in buffer that contains bovine serum albumin (below, referred to as the blocking buffer), and after this step of blocking, antibodies (first antibody) that bind with the desired protein are added to fresh blocking buffer, and the filter is immersed in this blocking buffer, so that the desired protein and the antibodies form complexes. After the filter is washed, it is immersed in a blocking buffer that contains the second antibody, labelled with an enzyme. The first antibody and the second antibody form complexes. Second antibodies coupled to alkaline phosphatase or peroxidase are commercially available. The filter is washed to remove excess second antibody that has not adsorbed to the filter, and the filter is put in a developing solution, and colored clones are selected. The recombinant phages obtained in this way are used to infect host Escherichia coli cells, and phage DNA is obtained from the phage lysate. The cDNA from the recombinant phage DNA is isolated and purified, and its DNA sequence is identified.
The antibodies used for immunological screening can be antisera prepared in rabbits immunized with other kinds of caldesmon, such as chicken caldesmon that have amino acid sequences likely to correspond in part to the sequence of the desired protein, human caldesmon.
The cDNA sequence coding for human caldesmon, which has been identified by the inventors, is sequence ID No.13 and 14 in the sequence listing. Of the two forms of human caldesmon, the 558 amino acid residues shown as sequence ID No.6 in the sequence listing is that of the caldesmon of high molecular weight, and the DNA sequence that codes for this is sequence ID No.12 of the sequence listing. The caldesmon of low molecular weight is exactly the same as that of high molecular weight except that it lacks amino acids 202 to 227, and it is shown as the 532 amino acids of sequence ID No.5 in the sequence listing, and the DNA sequence that codes for this is sequence ID No.11 in the sequence listing. Here, the form of higher molecular weight is called type I, and the form of lower molecular weight is called type II.
To obtain plasmids that express the polypeptides of human caldesmons type I and type II, DNA that codes for the caldesmon of type I and DNA that codes for the caldesmon of type II are prepared separetely from the recombinant phage DNA mentioned above, and the DNA is inserted into an expression plasmid, such as the plasmid pTV118N. The plasmid that coded for the caldesmon of type I was designated pTV118NHS3CaDl, and the plasmid that coded for the caldesmon of type II was designated pTV118NHS3CaD2.
With pTV118NHS3CaDl, it is possible to prepare DNA that codes for polypeptides of various lengths. For example, a site slightly upstream of the initiation codon of the region that codes for type I caldesmon of this plasmid can be cleaved with an appropriate restriction enzyme, and exonuclease can be used to remove the sequence of the 5'-side in the caldesmon gene. By changes in the reaction conditions, it is possible to prepare genes that code for polypeptides of various chain lengths that are missing the N-terminal portion of type I caldesmon. Alternatively, it is possible to amplify DNA of various lengths by the polymerase chain reaction (PCR: Saiki et al., Science, 230, 1350-1354, 1985) with a number of primers synthesized and pTV118NHS3CaD1 as a template, and said DNA can be used to prepare polypeptides of various lengths. As described in detail, with the sequence ID No.15 and 16 in the sequence listing as primers, PCR can be done (at 94°C for 30 sec in step 1, at 55°C for 2 min in step 2, and at 72°C for 1 min in step 3, for a total of 20 cycles), which results in the amplification of DNA that contains the DNA sequence shown as sequence ID No.10 in the sequence listing, which codes for the polypeptide shown in the sequence listing as having sequence ID No.4 (called 312AA below). With the sequence ID No.17 and 18 in the sequence listing as primers, it is possible to amplify DNA that contains the DNA sequence shown as sequence ID No.9 in the sequence listing, which codes for the polypeptide shown in the sequence listing as having sequence ID No.3 (called 122AA below). With the sequences ID No.18 and 19 in the sequence listing as primers, it is possible to amplify DNA that contains the DNA sequence shown as sequence ID No.8 in the sequence listing, which codes for the polypeptide shown in the sequence listing as having sequence ID No.2 (called 118AA below). With the sequence ID No.20 and 21 in the sequence listing as primer, it is possible to amplify DNA that contains the DNA sequence shown as sequence ID No.23 in the sequence list, which codes for the polypeptide shown in the sequence listing as having sequence ID No.22 (called 94AA below). In addition, with the sequences ID No.21 and 24 in the sequence listing as primers, it is possible to amplify DNA that contains the DNA sequence shown as sequence ID No.26 in the sequence listing, which codes for the polypeptide shown in the sequence listing as sequence ID No.25 (called 90AA below). Next, these DNAs are inserted into expression plasmids, such as, for example, pTV118N. The plasmid that coded for 312AA was named pTHCD247, the plasmid that coded for 122AA was named pTHCDXa443, the plasmid that coded for 118AA was named pTHCD443, the plasmid that coded for 94AA was named pTHCDXa451, and the plasmid that coded for 90AA was named pTHCD451.
The plasmids were used to transform Escherichia coli cells, such as E. coli JM109, for their expression, and the recombinant cells were cultured under appropriate conditions so that the desired polypeptides accumulated within the E. coli cells. The purification of the polypeptides was, for example, as follows. The recombinant E. coli cells were cultured in a culture medium such as L broth, and the cells were harvested and disrupted by being sonicated. The disrupted cells were centrifuged and the supernatant was obtained. After treatment such as dialysis, the dialysate was put on a column for gel filtration and then on a column for ion-exchange chromatography for purification. The desired proteins were purified by use of affinity columns coupled with calmodulin or tropomyosin, making use of the properties of the target protein. 122AA and 94AA have a sequence that can be recognized by factor Xa. By cleaving with factor Xa site-specifically, it is possible to prepare a polypeptide shown as sequence ID No.1 in the sequence listing (called 116AA below) and a polypeptide shown as sequence ID No.27 in the sequence listing (called 88AA below).
The polypeptides to which this invention is related are shown in Figure 2. Type II is a polypeptide that is missing one portion of type I. 312AA is a polypeptide that is the C-terminal portion of types I and II, 122AA and 118A are polypeptides that have a sequence of 116 amino acids in the C-terminal sequence of 312AA, and 94AA and 90AA are polypeptides that have a sequence of 88 amino acids that are in the central portion of 118AA, lacking both the N-terminal portion and the C-terminal portion. On the N-terminal of 118AA, there are residues Met-Ala that originated from a PCR primer, and on the N-terminal of 90AA, there are residues Met-Ala that also originated from a PCR primer. On the N-terminal of 122AA and 94AA, there are residues Met-Ala that originated from PCR primer and a sequence that can be recognized by factor Xa.

It is possible to measure the calmodulin-binding activity of these polypeptides by use of their affinity for calmodulin during calmodulinaffinity chromatography on a column, and it is possible to measure the actin-binding activity of these polypeptides by the method of coprecipitation with actin. As shown in Table 1, the polypeptide (typeI, typeII,312AA, 122AA, 118AA, and 116AA) that contains the polypeptide of 116 amino acids (called 116AA below) that has sequence ID No.1 in the sequence listing has both calmodulin-binding activity and actin-binding activity.

Table 1

polypeptide	calmodulin-binding activity	actin-binding activity
typeI	+	+
typeII	+	+
312AA	+	+
122AA	+	+
118AA	+	+
116AA	+	+
94AA	+	-
90AA	+	-
88AA	+	-
(Activity is shown as +, and lack of activity as -.)

Type I, type II, 312AA, 122AA, 118AA, 116AA all have tropomyosin-binding activity and have a inhibitory activity to actomyosin ATPase, and so the functional unit of human caldesmon is identified as being 116AA.
It is possible to construct a DNA that codes for 116AA out of pTHCD443 as needed by the method of site-specific mutagenesis, and the DNA can be used to express 116AA.
As described above in detail, this invention provides various polypeptides that have activities of human caldesmon and also provides genes that code for these polypeptides. It is possible to produce both on a large scale by the use of genetic engineering, and said genes and polypeptides will be useful in diagnosis and other fields of medicine, and in biochemical research, as well. The functional unit of human caldesmon, 116AA, can be used as a material for chimera proteins and peptide transfer, and the DNA with sequence ID No.7 in the sequence listing will be of use in the fields of genetic engineering and protein engineering.

Examples

Below, this invention will be explained in further detail with reference to examples, but this invention is not to be taken to be limited to these examples.

Example 1.

Cloning of cDNA that codes for the polypeptide of human caldesmon from HeLa cells

1-1. Construction of a cDNA library.

The total RNA of HeLa cells was obtained by the method of guanidium-cesium chloride (Biochemistry, 18, 5294-5299, 1979), and poly(A)⁺ RNA was isolated by column chromatography with oligo-(dT)cellulose. The purified poly(A)⁺ RNA was used as a template and oligo-(dT) was used as the primer in the synthesis of cDNA by the method of Gubler-Hoffmann (Gene, 25, 263-269, 1983). The ends of the cDNA were blunted with T4 DNA polymerase, and the EcoRI site in the cDNA was methylated with EcoRI methylase. Next, cDNA was ligated to an EcoRI linker [d(pGGAATTCC)] with use of T4 DNA ligase, and cDNA with both ends having an EcoRI was constructed by EcoRI digestion. This cDNA was ligated with the EcoRI arm of λgt11 (Stratagene), and a cDNA library was made by in vitro packaging with GigapackII Gold (Stratagene).

1-2. Screening of the cDNA library

Plaques of phages with use of E. coli Y1090 as the host cells were formed. To do this Y1090 cells were cultured overnight at 37°C in L medium that contained 0.02% maltose, and the cells were collected by centrifugation. The cells harvested were suspended in 10 mM MgSO₄ and were added into a suspension of phage solution and kept at 37°C for 15 minutes to allow attachment of the phages to the host cells. To this mixture was added a mixture of soft agar (L medium containing 10 mM MgSO₄ with agarose added to the final concentration of 0.6%, which medium was autoclaved and then cooled to and kept at 50°C), and this new mixture was spread on L plates (below, this step is referred to as "plating"). The plates were kept at 42°C for 3 hours, and after formation of plaques, a nylon membrane (Hybond-N; Amersham) treated with 100 mM IPTG was placed on the plate and the whole was kept at 37°C for 3 hours. The nylon membrane was removed from the plate and immnologically screened with rabbit anti-caldesmon antibodies obtained by the immunization of a rabbit with 35kDa fragment of chicken caldesmon obtained by the digestion, with α-chymotrypsin (J. Biochem., 102, 1065-1073, 1987) and with F(ab')₂ fragment of goat anti-rabbit immunoglobulin antibody labelled with alkaline phosphatase as the second antibody. Clones that were stained with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) were positive. Several positive signals were obtained from among the 250,000 clones. The plaques that corresponded to the positive signals were cut out together with agar and suspended in 500 µl of SM solution (50 mM Tris-HCl, 100 mM NaCl, and 10 mM MgSO₄, pH 7.5), plated after being diluted appropriately, and screened again as described above, by which procedure it was possible to isolate four phage clones independently.

1-3. Preparation of recombinant λgtl1 DNA.

Next, 10⁵ of the cloned phages were plated, and the plates were kept first at 42 °C for 3 hours and then at 37°C overnight. After this, 15 ml of SM solution and a few drops of chloroform were added and the plates were kept at room temperature for 30 minutes. The upper layer of soft agar with the SM solution was removed and put into a centrifuge tube, and centrifuged at 3000 r.p.m for 15 minutes. The supernatant was added 40% (w/v) polyethylene glycol 6000, so that the final concentration of the polyethylene glycol was 10%, and mixed throughly. The mixture was kept at 4°C for 1 hour and centrifuged at 3000 r.p.m. for 15 minutes. The supernatant was discarded and the precipitate was suspended with SM solution and then left at 4°C (below, this method is referred to as the plate lysate method). By this procedure, a phage solution with the titer of 1 x 10¹⁰ pfu/ml was obtained.
E. coli Y1090 cells that had been cultured overnight in L medium were harvested and suspended in 10 mM MgSO₄. The cell suspension was mixed with the phage solution mentioned above at multiplicity of infection (moi) of 0.01, and the mixture were left at 37°C for 15 minutes. The mixture of phages and E. coli cells was used to inoculate L medium that contained 10 mM MgSO₄, and continued to be cultured at 37°C until a considerable amount of bacterial debris remained after lysis. To the culture, sodium chloride was added to the final concentration of 0.5 M, chloroform was added to the final concentration of 0.5%, and the mixture was stirred at 37°C for 15 minutes. The supernatant obtained by centrifugation was mixed with polyethylene glycol 6000 added to the final concentration of 10% (w/v), and the mixture was left overnight at 4°C. The phage precipitate obtained by centrifugation was dissolved in TM solution (50 mM Tris-HCl and 10 mM MgSO₄, pH 7.8), and by the method of glycerol step-gradient ultracentrifugation (T.Maniastis et al., Molecular cloning: A laboratory manual, pp. 83-84, Cold Spring Harbor Laboratory, 1982), the phages were purified.
The phages obtained were suspended in TM solution, and DNase I and RNaseA were added to the suspension, which was then kept at 37°C for 30 minutes before the addition of EDTA, Proteinase K (Sigma), and SDS to the concentrations of 20 mM 50 µg/ml, and 0.5%, respectively. After then the mixture was kept at 65°C for 1 hour. Phenol extraction was done, followed by extraction with diethyl ether, and to the aqueous layer, a 1/10 volume of 5 M sodium chloride and two volumes of ice-cold ethanol were added, which caused precipitation of the DNA (below, this step is referred to as ethanol precipitation). The mixture was centrifuged and the precipitated DNA was collected, after which it was washed in 70% ethanol and dried before being dissolved in 100 µl of TE solution (10 mM Tris-HCl and 1 mM EDTA, pH 8.0).

1-4. Identification of DNA sequence of the inserted fragment

The recombinant λgt11 DNA prepared as described above was digested with EcoRI, and the inserted fragments were isolated and purified. Then they were cloned at the EcoRI site of M13mp18RF DNA. The recombinant M13mp18RF DNA was digested with SalI and SphI, and by use of exonuclease III (Course in Biochemical Experimentation 1, Methods in genetic research I, pp. 186-200, 1986), a mutant was constructed that lacked various length of sequence at the 5'-side of the SalI end. Another deletion mutant was prepared that was of recombinant M13mp18RF DNA that had the fragment insertion in the opposite orientation. Then E. coli JM109 cells were transformed with each mutant DNA derived from the recombinant M13mp18 and single-stranded DNA was isolated from the M13mp18 derivatives. Its DNA sequence was identified by the dideoxy-mediated chain-termination method, and the amino acid sequence was deduced from the DNA sequence.
Restriction maps of these clones are shown in Fig. 1. The ATG and TGA shown in the Fig. 1 are the initiation codon and the termination codon for translation, respectively.
The results of identification of the DNA sequence suggested that HeLa cells had two kinds of caldesmon. This was checked by use of the PCR. To do this, with primer 6 (shown in Fig. 1 as P6) indicated as sequence ID No.29 in the sequence listing the first strand of cDNA was synthesized. With both primer 5 (shown in Fig. 1 as P5) indicated as sequence ID No.30 of the sequence listing and primer 4 (shown in Fig. 1 as P4) indicated as sequence ID No.31 in the sequence listing as primer 4, the PCR was done, and two kinds of PCR product A (A-1 and A-2) were obtained. Then with primer 5 and primer 28 (shown in Fig. 1 as P28) indicated as sequence ID No.32, the PCR was done, and two kinds of PCR product B (B-1 and B-2) were obtained. Restriction maps of PCR products A (A-1 and A-2) and B (B-1 and B-2) are shown in Fig. 1. The DNA sequences of these PCR products were identified by the dideoxy method, and it was confirmed that there were two kinds of caldesmon. The caldesmon of higher molecular weight was composed of the 558 amino acid residues shown as sequence ID No.6 in the sequence listing, and the caldesmon of lower molecular weight was composed of the 532 amino acid residues shown as sequence ID No.5 in the sequence listing and was exactly the same as that of higher molecular weight except that it lacked amino acid residues 202 to 227 of the larger caldesmon. The caldesmon of higher molecular weight was named type I, and the caldesmon of lower molecular weight was named type II. The DNA sequence of type I is that shown as sequence ID No.12 in the sequence listing, and the DNA sequence of type II is that shown as sequence ID No.11. Their cDNA sequences are shown as sequence ID No.14 and 13, respectively, in the sequence listing.

Example 2. Construction of expression vectors for types I and II

2-1. Construction of a plasmid that coded for type I caldesmon.

A cDNA fragment of about 800 bp that coded for the N-terminal region of type I caldesmon was obtained by EcoRI digestion of λgt11HS3CaDN1, and cloned at the EcoRI site of M13mp18RF DNA, giving M13mp18HS3CaDN1. This M13mp18HS3CaDN1 was digested with Nsp(7524)I, blunt-ended with T4 DNA polymerase, and digested with EcoRI, giving cDNA fragments of about 790 bp. This cDNA fragment was cloned in plasmid pTV118N which was digested with NcoI, treated with Klenow fragment (large fragment of E. coli DNA polymerase I), and digested with EcoRI, giving pTV118NHS3CaDN1.
cDNA fragments about 1.3 kbp long that coded for the C-terminal region of caldesmon were obtained by EcoRI digestion of λgt11HS3CaD25, and cloned at the EcoRI site of M13mp18RF DNA, giving M13mp18HS3CaDC. Then M13mp18HS3CaDC was digested with EcoRI and SacI, giving fragments of cDNA about 940 bp long, which were into pTV118NHS3CaDN1 between the EcoRI and SacI sites , giving plasmid pTV118NHS3CaD1, which coded for type I caldesmon.
Cells of E. coli JM109 into which pTV118NHS3CaD1 had been introduced were designated E. coli JM109/pTV118NHS3CaD1, and deposited at the Fermentation Research Institute of the Agency of Industrial Science and Technology, Japan, under FERM BP-3673.

2-2. Construction of a plasmid that coded for type II caldesmon

T4 DNA polymerase was used to make blunt ends on PCR product A-2, and the product was cloned at the SmaI site of M13mp18RF DNA so as to be in the same direction in the PCR product A-2 as in the M13mp18 lacZ gene. Then cloned M13mp18PCRA-2 with the plasmid A-2 as an insertion was constructed. M13mpl8PCRA-2 was digested with Nsp(7524)I, and after its ends were made blunt with T4 DNA polymerase, it was digested at the multicloning site that originated from M13mp18 with XbaI, and cDNA fragments about 1.6 kbp long were obtained. These were cloned into pTV118N that had been digested with NcoI, treated with Klenow fragment, and then digested with XbaI, giving pTV118NHS3CaD2 PCRA-2. This was digested with EcoRI and HindIII, and ligated with the cDNA fragments about 940 bp long that code for the C-terminal region of the caldesmon obtained by digestion of pTV118NHS3CaD1 with EcoRI and HindIII. The product was designated plasmid pTV118NHS3CaD2, which codes for type II caldesmon.
E. coli cells into which pTV118NHS3CaD2 had been introduced were designated E. coli JM109/pTV118NHS3CaD2, and deposited at the Fermentation Research Institute of the Agency of Industrial Science and Technology, Japan, under FERM P-12013.

Example 3. Construction of a plasmid that expresses human caldesmon polypeptide

3-1. Construction of a plasmid that codes for 312AA

A primer with sequence ID No.15 of the sequence listing and with the NcoI recognition sequence at its 5'-end and a primer with sequence ID No.16 and with the SacI recognition sequence at its 5'-end were synthesized with a DNA synthesizer and purified. Said primers were used together with pTV118NHS3CaDl as the template in the PCR to amplify DNA that had the DNA sequence shown as sequence ID No.10 in the sequence listing within its sequence.
Next, this amplification product was digested with NcoI and SacI and extracted with buffered phenol, so that its enzyme activity was lost. For the removal of an excess of dNTP in the PCR reaction mixture, and DNA concentration, the DNA was caused to precipitate with ammonium sulfate and isopropyl alcohol. The precipitated DNA was dissolved with TE solution (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA), and ligated with pTV118N that had been digested with both NcoI and SacI, giving the plasmid pTHCD247, which codes for 312AA.
E. coli JM109 cells into which pTHCD247 had been introduced were designated E. coli JM109/pTHCD247, and deposited at the Fermentation Research Institute of the Agency of Industrial Science and Technology, Japan, under FERM BP-3671.

3-2. Construction of a plasmid that codes for 118AA

A primer with sequence ID No.19 of the sequence listing and with the NcoI recognition sequence at its 5'-end and a primer with sequence ID No.18 and with the EcoRI recognition sequence at its 5'-end were synthesized with a DNA synthesizer and purified. Said primers were used together with pTV118NHS3CaD1 as the template in the PCR to amplify DNA that had the DNA sequence shown as sequence ID No.8 in the sequence listing within its sequence.
Next, this amplification product was digested with NcoI and EcoRI, and treated as in section 3-1 above before being ligated with pTV118N that had been digested with both NcoI and EcoRI, giving the plasmid pTHCD443, which codes for 118AA.
E. coli JM109 cells into which pTHCD443 had been introduced were designated E. coli JM109/pTHCD443, and deposited at the Fermentation Research Institute of the Agency of Industrial Science and Technology, Japan, under FERM BP-3672.

3-3. Construction of a plasmid that codes for 122AA

A primer with sequence ID No.17 of the sequence listing and with the NcoI recognition sequence and the factor Xa recognition sequence at its 5'-end was synthesized with a DNA synthesizer and purified. Next, said primer and the primer described above as having sequence ID No.18 were used together with pTV118NHS3CaD1 as the template in the PCR to amplify DNA that had the DNA sequence shown as sequence ID No.9 in the sequence listing within its sequence.
Next, this amplification product was digested with NcoI and EcoRI, and ligated with pTV118N, which had also been digested both NcoI and EcoRI, giving a plasmid that coded for 122AA. Said plasmid was designated pTHCDXa443, and cells of E. coli JM109 into which the plasmid was introduced were named E. coli JM109/pTHCDXa443.

3-4. Construction of a plasmid that codes for 90AA

A primer with sequence ID No.24 of the sequence listing and with the NcoI recognition sequence at its 5'-end and a primer with sequence ID No.21 and the EcoRI recognition sequence at its 5'-end were synthesized with a DNA synthesizer and purified. Said primers were used together with pTV118NHS3CaD1 as the template in the PCR to amplify DNA that had the DNA sequence shown as sequence ID No.26 in the sequence listing within it. Next, this amplification product was digested with NcoI and EcoRI, and ligated with pTV118N that had been digested with NcoI and EcoRI, giving the plasmid pTHCD451, which codes for 90AA.
E. coli JM109 cells into which pTHCD451 had been introduced were designated E. coli JM109/pTHCD451.

3-5. Construction of a plasmid that codes for 94AA

A primer with sequence ID No.20 of the sequence listing and with the NcoI recognition sequence and the factor Xa recognition sequence at its 5'-end was synthesized with a DNA synthesizer and purified. Said primer and the primer described above as having sequence ID No.21 were used together with pTV118NHS3CaD1 as the template in the PCR to amplify DNA that had the DNA sequence shown as sequence ID No.23 in the sequence listing within its sequence.
Next, this amplification product was digested with NcoI and EcoRI, and ligated with pTV118N, which had also been digested both NcoI and EcoRI, giving a plasmid that coded for 94AA. Said plasmid was designated pTHCDXa451, and cells of E. coli JM109 into which the plasmid was introduced were named E. coli JM109/pTHCDXa451.

Example 4. Expression of human caldesmon polypeptide in E. coli

4-1. Expression of type I in E. coli

Cells of E. coli JM109 which carried pTV118NHS3CaD1 (FERM BP-3673) were used to inoculate 5 ml of L medium that contained 50 µg/ml ampicillin, and the culture was incubated with shaking overnight at 37°C. This seed culture was used to inoculate 500 ml of L medium with the same concentration of ampicillin, and the culture was incubated with shaking overnight at 37°C. When the cells reached the mid-logarithmic stage of growth, IPTG was added to the final concentration of 2 mM, and culture was continued for 17 hours more before the cells were harvested.
The cells were suspended in E solution (0.3 M KCl, 0.5 mM EGTA, 0.5 mM MgC1₂, 0.5 mM dithiothreitol (DTT), 0.3 mM PMSF, and 50 mM Tris-HCl, pH 7.0), and the suspension was sonicated to disrupt the cells. The suspension was centrifuged and the supernatant was incubated at 95°C for 5 minutes and then cooled. This was centrifuged and the precipitate was removed. The supernatant was dialyzed against C1 solution (120 mM NaCl, 0.1 mM EGTA, 0.2 mM DTT, and 10 mM Tris-HCl, pH 7.3). The inner solution during dialysis was passed through a column of DEAE Toyopearl 650M equilibrated with C1 solution. To the pass-through fraction, calcium chloride was added to the final concentration of 5 mM, the mixture was passed through a column of Sepharose 4B (Pharmacia LKB) coupled with calmodulin which equilibrated with C2 solution (70 mM NaCl, 0.2 mM CaCl₂, 0.1 mM DTT, and 10 mM Tris-HCl, pH 7.5) as described elsewhere (Journal of Biochemistry, 102, 1065-1073, 1987), and type I purified was extracted with C2 solution that contained 1.2 mM EGTA and purified.

4-2. Expression of type II in E. coli

Cells of E. coli JM109 which carried pTV118NHS3CaD2 (FERM P-12013) were cultured under the same conditions as in the example of 4-1 above, extracted, heated, put on a column of resin for affinity chromatography with calmodulin, and purified, giving type II.

4-3. Expression of 312AA, 122AA, 118AA, 94AA, and 90AA in E. coli

Cells of E. coli JM109/pTHCD247 (FERM BP-3671), of E. coli JM109/pTHCDXa443, of E. coli JM109/pTHCD443 (FERM BP-3672), of E. coli JM109/pTHCDXa451 and of E. coli JM109/pTHCD451 were cultured under the same conditions as in the example of 4-1 above, and purification gave 312AA, 122AA, 118AA, 94AA, and 90AA.

4-4. Preparation of 116AA and 88AA

122AA and 94AA were treated with restriction proteinase factor Xa (Takara Shuzo Co., Ltd.) for 5 hours at 37°C, and a column of calmodulin-Sepharose was used to purify 116AA and 88AA from the digest.

Example 5. Assay of physiological activity

5-1. Assay of calmodulin-binding activity and actin-binding activity

For the assay of calmodulin-binding activity, polypeptides purified as described above were mixed with 10 volumes of CaB buffer (10 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM DTT, and 0.2 mM CaC1₂), and put on a column of Sepharose 4B (Pharmacia) coupled with calmodulin which equilibrated with the same buffer. This allowed binding of the polypeptide with calmodulin. After the column was washed with a large amount of the same buffer, the bound polypeptide was eluted from the column with the same buffer, except that 1 mM EGTA was added to the buffer instead of CaC1₂. A portion of each fraction was put on an SDS-polyacrylamide gel and electrophoresed, and the presence of the polypeptide in question was confirmed. For the assay of actin-binding activity, the co-precipitation method (BBRC, 132, 645-651, 1985, and Journal of Biochemistry, 102, 1065-1073, 1987) was used.
To 100µ1 of a 2 mg/ml solution of monomeric actin prepared by the Staub method (Journal of Biological Chemistry, 188, 559 (1959)), 20 µl of 10 mM ATP, 40 µl of 5x buffer (50 mM Tris-HCl, pH 7.5, 500 mM KCl, 1 mM CaC1₂, and 0.5 mM DTT), 20 µl purified polypeptide, and water were added for a total volume of 196 µl, and then 4 µl of 100 mM MgC1₂ was added. The mixture was left at room temperature for 1 hour. By the addition of MgC1₂, the monomeric actin was polymerized and became filamentous actin. After the polymerization, the mixture was centrifuged at 200,000 x g for 30 minutes. The monomeric actin remained in the supernatant, whereas the filamentous actin and proteins bound with it (the polypeptide of this invention) precipitated together. The supernatant and the precipitate were separately electrophoresed on SDS-polyacrylamide gels, and the binding of the polypeptide with actin was confirmed.
The results shown in the table 1 above, were that the polypeptide that contained 116AA in its sequence had both kinds of activity.

5-2. Assay of tropomyosin-binding activity and inhibition of actomyosin ATPase activity

The ability of type I, type II, 312AA, 122AA, 118AA, and 116AA which have the activities described in 5-1, to bind with tropomyosin and to inhibit actomyosin ATPase activity was measured.
For the assay of tropomyosin-binding activity, an affinity column with tropomyosin was prepared by the coupling of tropomyosin with Sepharose 4B (Pharmacia) activated with CNBr. The column was equilibrated with TMB buffer (10 mM Tris-HCl, pH 7.0, 2 mM MgC1₂, and 0.5 mM DTT), and a solution of a purified polypeptide diluted 10 times in the same buffer was put on the column to allow binding of the polypeptide to the column. The column was washed throughly with the same buffer, and after substances that had bound nonspecifically were removed in this way, elution was done with slowly increasing concentrations of potassium chloride. A portion of each fraction was put on an SDS-polyacrylamide gel and electrophoresed, and the polypeptide in question was found in the fractions that had been eluted with potassium chloride at the concentrations of 40 to 60 mM.
For the assay of the inhibition of actomyosin-ATPase by the polypeptide of this invention, first, phosphorylated myosin (final concentration, 0.2 mg/ml), actin (final concentration, 0.1 mg/ml), and tropomyosin (final concentration, 30 µg/ml) were added to a buffer that contained 20 mM imidazole-HCl (pH 7.2), 0.1 mM DTT, 100 mM KCl, 2 mM MgC1₂, 1 mM ATP, and 0.1 mM CaC1₂. Then the purified polypeptide was added and the mixture was kept at 30°C for 5 minutes. After this reaction time, trichloroacetic acid was added to the final concentration of 10% to stop the reaction. The reaction mixture was centrifuged, and the amount of free phosphate in the supernatant was assayed by the method of Youngburg (BBRC, 132, 645-651, 1985). The inhibition caused by the polypeptide was found from the difference in the amount of free phosphate related by ATPase in the presence and absence of the polypeptide. Both of these activities were found in type I, type II, 312AA, 122AA, 118AA, and 116AA. The functional unit of human caldesmon polypeptide was found to be 116AA.

Results of the invention.

As shown by the results described above, this invention provides the complete amino acid sequence of human caldesmon, and the complete DNA sequence that codes for human caldesmon, and it identifies the functional unit of caldesmon activity. These genes and the polypeptides are useful in the fields of biochemistry, medicine, including diagnosis, and the like.

Explanation of the figures.

Figure 1 is a restriction map of the cDNA that codes for type I and type II caldesmon (CaD). Figure 2 is a figure that shows the relationships of type I, type II, 312AA, 118AA, and 90AA.

Claims

An isolated and non-naturally occurring polypeptide having calmodulin-binding activity and actin-binding activity and comprising the amino acid sequence shown in SEQ ID No 1 in a polypeptide molecule.
An isolated and non-naturally occurring polypeptide according to claim 1 having the amino acid sequence shown in SEQ ID No. 1, SEQ ID No 2, SEQ ID No. 3 or SEQ ID No. 4.
An isolated gene which codes for a polypeptide according to claim 1 or 2.
An isolated gene according to claim 3 comprising the DNA sequence shown in SEQ ID No. 7 in a gene molecule.
An isolated gene according to claim 4 having the DNA sequence shown in SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 or SEQ ID No. 10.
An isolated gene according to claim 3 which can hybridise the gene of claim 4 or claim 5 under stringent conditions.